**"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures**

Letizia Marsili, Silvia Maltese, Daniele Coppola, Ilaria Caliani, Laura Carletti, Matteo Giannetti, Tommaso Campani, Matteo Baini, Cristina Panti, Silvia Casini and M. Cristina Fossi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54429

**1. Introduction** 

Cetacean diversity, like all biodiversity worldwide, is seriously threatened; its loss seems to be occurring at a very rapid and increasing rate [1]. In March 2010, the European Commission set a key objective for 2020: halt the loss of biodiversity and the degradation of ecosystem services in the EU [2]. To the objective of improving the effectiveness of conservation strategies it becomes important to know the health status of endangered species and then to develop methods of investigation that are not destructive and the least invasive possible. In the last few years a non destructive sampling method, the skin biopsy, was developed in cetaceans to obtain viable tissue samples from free-ranging animals [3]. With the skin biopsy it is possible to assess the effects of multiple pressures related to bioaccumulation of anthropogenic contaminants, infectious diseases, climate change, food depletion from over-fishing, bycatch, noise, shipping and collision that stress cetacean species. The evaluation of their "health status" is possible using a suite of sensitive tools, such as non-destructive biomarkers, that will enable us to detect the presence and the effects of contaminants, the reproduction alteration, the genotoxicity, the immunosuppression, the feeding ecology and the general stress [4]. Actually, it is very difficult to discern the effects of one threat from those of another when multiple threats are acting simultaneously; for example the incidence of pathology in cetaceans is closely related to the level of pollution in their environments and thus bacterial and viral infections and contaminants should be considered from a holistic point of view [5]. Regarding the effects of anthropogenic

© 2012 Marsili et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

contaminants, it is known that some contaminants, such as organochlorines (OCs), polybrominated diphenyl ethers (PBDEs), bisphenol A (BPA) and phthalates are endocrine disrupting chemicals (EDCs) and immunosuppressors [6, 7, 8, 9, 10]. Others, such as polycyclic aromatic hydrocarbons (PAHs), derived from both natural (e.g., oil spills, forest fires, natural petroleum seeps) and anthropogenic (e.g., combustion of fossil fuels, use of oil for cooking and heating, coal burning) sources, are carcinogenic, teratogenic and mutagenic compounds [11] and some studies have shown that PAHs with four or more rings can induce dioxin-like activity and weak estrogenic responses [12]. Moreover PAHs have attracted scientific interest due to their genotoxicity [13]. But how is it possible to discriminate the effects of a specific toxic in a mixture of many pollutants and assess the susceptibility of a particular cetacean species to just one class of contaminants? The aim of the present study is to use cetacean fibroblast cell cultures, obtained from skin biopsy of free-ranging animals and from skin tissue of stranded animals dead within 12 h [14, 15], as an "in vitro" method, called "*Test Tube Cetaceans*", to investigate the effects of environmental contaminants. In particular we use *Test Tube Cetaceans* to explore the susceptibility to genotoxic effects of different environmental contaminants in these marine mammals. Cell cultures were obtained from several species of cetaceans: fin whale (*Balaenoptera physalus*) and Bryde's whale (*Balaenoptera edeni*) for mysticetes, sperm whale (*Physeter macrocephalus*), killer whale (*Orcinus orca*), Risso's dolphin (*Grampus griseus*), bottlenose dolphin (*Tursiops truncatus*), striped dolphin (*Stenella coeruleoalba*), long-beaked common dolphin (*Delphinus capensis*) and common dolphin (*Delphinus delphis*) for odontocetes. Here we present the results for three different biomarkers of anthropogenic stress in cetacean cell cultures that will enable us to assess: i) exposure to contaminants, ii) immunosuppression and iii) genotoxicity.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 51

endothelial cells and fibroblasts. MIC molecules are considered to be stress-induced antigens that are recognized by cytotoxic T cells and natural killer (NK) cells, which play an important role in the surveillance of transformed infected and damaged cells [20]. Because the cetacean skin is an important tissue of the immune system and contributes to biological structure by acting not only as a protective physical barrier, but also as a target for immune components that mount the initial defense against invading pathogens, noxious stimuli, and resident neoplastic cells, the evaluation of MICA protein expression in cetaceans can be used to evaluate the status of the immune system

iii. **Detection of DNA damage by Comet assay as genotoxicity biomarker:** the presence of compounds such as PAHs, OCs and heavy metals in the marine environment can damage the DNA of living cells. The loss of DNA integrity can determine genotoxic effects, such as DNA base modifications, strand breaks, depurination and cross-linkages [21]. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method used as an indicator of genotoxicity and an effective biomarker for detecting DNA damage in living cells of aquatic animals [22]. Compared to other genotoxicity tests, such as chromosomal aberrations, sister chromatid exchanges, alkaline elution, and micronucleus assay, the advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage (one break per 1010 Da of DNA), requirement for small number of cells (~10,0000) per sample, flexibility to use proliferating cells, low cost, ease of

Samples of skin (epidermis, dermis and blubber) were obtained from free-ranging specimens of long-beaked common dolphin (*Delphinus capensis*; MDC12) and common dolphin (*Delphinus delphis*; DDL1) using an aluminium pole armed with biopsy tips (0.7 cm ø, 3.0 cm length), while skin biopsies from free-ranging specimens of Bryde's whale (*Balaenoptera edeni*; MBE3), killer whale (*Orcinus orca*; MOO12), sperm whale (*Physeter macrocephalus*; PMAS1), bottlenose dolphin (*Tursiops truncatus*; TTA1) and Risso's dolphin (*Grampus griseus*; GGL1) were obtained using a Barnett Wildcat II crossbow with a 150 pound test bow, using a biopsy dart with modified stainless steel collecting tip (0.9 cm ø, 4.0 cm length). Biopsy samples were taken in the dorsal area near the dorsal fin, with CITES authorization (CITES Nat. IT025IS, Int. CITES IT 007) in the Sea of Cortez (MDC12, MBE3 and MOO12) and Mediterranean Sea (DDL1, PMAS1, TTA1 and GGL1). A small fragment of

Skin tissue of stranded cetaceans (dead within 2-12 h) were obtained from specimens found dead along the Italian coasts in the period 2005–2009 (CITES Nat. IT025IS, Int. CITES IT 007). Samples were taken under the dorsal fin of stranded specimens of fin whale (RT2 and

application, and the short time needed to complete a study [23].

the biopsy was immediately stored in cell medium for the cell cultures.

of different species of cetaceans.

**2. Sampling methods** 

**2.2. Stranded cetaceans** 

**2.1. Free- ranging cetaceans** 


endothelial cells and fibroblasts. MIC molecules are considered to be stress-induced antigens that are recognized by cytotoxic T cells and natural killer (NK) cells, which play an important role in the surveillance of transformed infected and damaged cells [20]. Because the cetacean skin is an important tissue of the immune system and contributes to biological structure by acting not only as a protective physical barrier, but also as a target for immune components that mount the initial defense against invading pathogens, noxious stimuli, and resident neoplastic cells, the evaluation of MICA protein expression in cetaceans can be used to evaluate the status of the immune system of different species of cetaceans.

iii. **Detection of DNA damage by Comet assay as genotoxicity biomarker:** the presence of compounds such as PAHs, OCs and heavy metals in the marine environment can damage the DNA of living cells. The loss of DNA integrity can determine genotoxic effects, such as DNA base modifications, strand breaks, depurination and cross-linkages [21]. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method used as an indicator of genotoxicity and an effective biomarker for detecting DNA damage in living cells of aquatic animals [22]. Compared to other genotoxicity tests, such as chromosomal aberrations, sister chromatid exchanges, alkaline elution, and micronucleus assay, the advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage (one break per 1010 Da of DNA), requirement for small number of cells (~10,0000) per sample, flexibility to use proliferating cells, low cost, ease of application, and the short time needed to complete a study [23].
